A network of acetyl phosphate-dependent modification modulates c-di-AMP homeostasis in Actinobacteria.

Cyclic purine nucleotides are important signal transduction molecules across all domains of life. 3',5'-cyclic di-adenosine monophosphate (c-di-AMP) has roles in both prokaryotes and eukaryotes, while the signals that adjust intracellular c-di-AMP and the molecular machinery enabling a network-wide homeostatic response remain largely unknown. Here, we present evidence for an acetyl phosphate (AcP)-governed network responsible for c-di-AMP homeostasis through two distinct substrates, the diadenylate cyclase DNA integrity scanning protein (DisA) and its newly identified transcriptional repressor, DasR. Correspondingly, we found that AcP-induced acetylation exerts these regulatory actions by disrupting protein multimerization, thus impairing c-di-AMP synthesis via K66 acetylation of DisA. Conversely, the transcriptional inhibition of disA was relieved during DasR acetylation at K78. These findings establish a pivotal physiological role for AcP as a mediator to balance c-di-AMP homeostasis. Further studies revealed that acetylated DisA and DasR undergo conformational changes that play crucial roles in differentiation. Considering the broad distribution of AcP-induced acetylation in response to environmental stress, as well as the high conservation of the identified key sites, we propose that this unique regulation of c-di-AMP homeostasis may constitute a fundamental property of central circuits in Actinobacteria and thus the global control of cellular physiology.IMPORTANCESince the identification of c-di-AMP is required for bacterial growth and cellular physiology, a major challenge is the cell signals and stimuli that feed into the decision-making process of c-di-AMP concentration and how that information is integrated into the regulatory pathways. Using the bacterium Saccharopolyspora erythraea as a model, we established that AcP-dependent acetylation of the diadenylate cyclase DisA and its newly identified transcriptional repressor DasR is involved in coordinating environmental and intracellular signals, which are crucial for c-di-AMP homeostasis. Specifically, DisA acetylated at K66 directly inactivates its diadenylate cyclase activity, hence the production of c-di-AMP, whereas DasR acetylation at K78 leads to increased disA expression and c-di-AMP levels. Thus, AcP represents an essential molecular switch in c-di-AMP maintenance, responding to environmental changes and possibly hampering efficient development. Therefore, AcP-mediated posttranslational processes constitute a network beyond the usual and well-characterized synthetase/hydrolase governing c-di-AMP homeostasis.

Beneficial effects of American ginseng (Panax quinquefolius L.) extract residue as a feed additive on production, health status, and gastrointestinal bacteria in sika deer (Cervus nippon).

American ginseng residue is an industrial by-product of ginseng saponin extraction, including polysaccharides and amino acids; however, it is often discarded into the natural environment, representing a waste of resources as well as an environmental issue. In this study, we examined the effects of adding American ginseng residue to the basal diet of sika deer. Twelve antler-bearing male sika deer were assigned randomly to groups fed a diet supplemented with 0% (CON), 1% (LGR), and 3% (HGR) American ginseng residue, respectively, (n = 4 per group) for 5 weeks. Supplementation with 3% American ginseng residue significantly increased antler production and feed utilization efficiency in antler-bearing sika deer (p < 0.05). There were no significant differences in serum biochemical indexes among the three groups, but serum immunoglobulin A and glutathione peroxidase levels were significantly increased in the LGR and HGR groups (p < 0.05). Supplementation with American ginseng residue affected rumen fermentation in sika deer, significantly increasing the rumen contents of acetic acid, propionic acid, and total volatile fatty acids, and decreasing rumen fluid pH (p < 0.05), but had no significant effect on microbial protein or ammoniacal nitrogen content. American ginseng residue also affected the rumen bacterial composition, with significant up-regulation of Bacteroidota abundance in the HGR group, significant increases in Fibrobacterota and Fibrobacter abundance in the LGR group, and a significant decrease in Oscillospiraceae_UCG-005. Supplementation with ginseng residue had no significant effect on volatile fatty acids in the feces of sika deer, but did affect the composition of fecal bacteria, with significant decreases in Desulfobacterota and Rikenellaceae_RC9_gut_group in the HGR group, and a significant increase in Ruminococcus in the LGR group (p < 0.05). In addition, the abundance of Paeniclostridium in the feces decreased linearly with increasing concentration of ginseng residue, with a significant difference among the groups (p < 0.05). This study comprehensively evaluated the effects of American ginseng residue as a potential feed additive on the production performance and gastrointestinal bacterial community in antler-bearing sika deer. The results indicated that ginseng residue was a suitable feed additive for improving production performance and health in sika deer.

Self-amplified activatable nanophotosensitizers for HIF-1α inhibition-enhanced photodynamic therapy.

Activatable photodynamic therapy (PDT) has shown great potential in cancer therapy owing to its high tumor specificity and minimized side effect. However, the relatively low level of biomarkers within tumor tissue rescricts the photosensitizer to get thoroughly activated. In this study, we design a self-amplified activatable nanophotosensitizer (CPPa NP) for enhanced PDT. CPPa NP is prepared by encapsulating a hypoxia-inducible factor 1α (HIF-1α) inhibitor CI-994 with an amphiphilic hydrogen peroxide (H2O2) responsive copolymer PPa-CA-PEG. Upon the addition of H2O2, the thioketal linker within CPPa NP is cleaved, resulting in the simultaneous release of thiol-modified pyropheophorbide a (PPa-SH), cinnamic aldehyde (CA), and CI-994. PPa-SH can be encapsulated by albumin to turn on its photodynamic efficiency, while CI-994 may inhibit the expression of HIF-1α to improve the PDT efficacy. CA is able to deplete glutathione (GSH) and upregulate reactive oxygen species (ROS) within tumor cells, accelerating the dissociation of nanoparticles and disrupting the redox balance of tumor cells. In vitro and in vivo studies showed that CPPa NP can successfully elevate the ROS level within 4T1 cells and has a better anticancer efficacy than PPa NP without CI-994 under laser irradiation. This study thus provides an effective approach to develop self-amplified activatable nanoparticles for enhanced PDT.

Prolonging Treatment Window of Photodynamic Therapy with Self-Amplified H2 O2 -Activated Photodynamic/Chemo Combination Therapeutic Nanomedicines.

Photodynamic therapy (PDT) is a promising approach to cancer therapy. However, the relatively short tumor retention time of photosensitizers (PSs) makes it difficult to catch the optimal treatment time and restricts multiple PDT within a single injection. In this study, a tumor-specific phototheranostic nanomedicine (DPPa NP) is developed for photodynamic/chemo combination therapy with a prolonged PDT treatment window. DPPa NP is prepared via encapsulating a hydrophobic oxidized bovine serum albumin (BSA-SOH)-conjugatable PS DPPa with amphiphilic H2 O2 -activatable chlorambucil (CL) prodrug mPEG-TK-CL. The released CL under H2 O2 treatment can not only kill tumor cells but also upregulate reactive oxygen species levels within tumor cells, leading to the almost full release of cargoes. The released DPPa may conjugate with overexpressed BSA-SOH, which results in the recovery of the fluorescence signal and photodynamic effect. More importantly, such conjugation transfers DPPa from a small molecule PS into a macromolecular PS with a long tumor retention time and treatment window of PDT, which enables multiple PDT. This study thus provides an effective strategy to prolong the treatment window of PDT and enables tumor-specific fluorescence imaging-guided combination therapy.

A meet-up of acetyl phosphate and c-di-GMP modulates BldD activity for development and antibiotic production.

Actinobacteria are ubiquitous bacteria undergoing complex developmental transitions coinciding with antibiotic production in response to stress or nutrient starvation. This transition is mainly controlled by the interaction between the second messenger c-di-GMP and the master repressor BldD. To date, the upstream factors and the global signal networks that regulate these intriguing cell biological processes remain unknown. In Saccharopolyspora erythraea, we found that acetyl phosphate (AcP) accumulation resulting from environmental nitrogen stress participated in the regulation of BldD activity through cooperation with c-di-GMP. AcP-induced acetylation of BldD at K11 caused the BldD dimer to fall apart and dissociate from the target DNA and disrupted the signal transduction of c-di-GMP, thus governing both developmental transition and antibiotic production. Additionally, practical mutation of BldDK11R bypassing acetylation regulation could enhance the positive effect of BldD on antibiotic production. The study of AcP-dependent acetylation is usually confined to the control of enzyme activity. Our finding represents an entirely different role of the covalent modification caused by AcP, which integrated with c-di-GMP signal in modulating the activity of BldD for development and antibiotic production, coping with environmental stress. This coherent regulatory network might be widespread across actinobacteria, thus has broad implications.

AS-605240 Blunts Osteoporosis by Inhibition of Bone Resorption.

Background: Osteoporosis is a metabolic bone disease. Osteoclasts are significantly involved in the pathogenesis of osteoporosis. AS-605240 (AS) is a small molecule PI3K-γ inhibitor and is less toxic compared to pan-PI3K inhibitors. AS also exerts multiple biological effects including anti-inflammatory, anti-tumor, and myocardial remodeling promotion. However, the involvement of AS in the differentiation and functions of osteoclasts and the effect of AS in treating patients with osteoporosis is still unclear. Purpose: This study aimed to investigate if AS inhibits the differentiation of osteoclasts and resorption of the bones induced by M-CSF and RANKL. Next, we evaluated the therapeutic effects of AS on bone loss in ovariectomy (OVX)-induced osteoporosis mice models. Methods: We stimulated bone marrow-derived macrophages with an osteoclast differentiation medium containing different AS concentrations for 6 days or 5µM AS at different times. Next, we performed tartrate-resistant acid phosphatase (TRAP) staining, bone resorption assay, F-actin ring fluorescence, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting (WB). Next, MC3T3-E1s (pre-osteoblast cells) were differentiated to osteoblast by stimulating the cells with varying AS concentrations. Next, we performed alkaline phosphatase (ALP) staining, RT-qPCR, and WB on these cells. We established an OVX-induced osteoporosis mice model and treated the mice with 20mg/kg of AS. Finally, we extracted the femurs and performed micro-CT scanning, H&E, and TRAP staining. Results: AS inhibits the formation of osteoclasts and resorption of bone triggered by RANKL by inhibiting the PI3K/Akt signaling pathway. Furthermore, AS enhances the differentiation of osteoblasts and inhibits bone loss due to OVX in vivo. Conclusion: AS inhibits osteoclast production and enhances osteoblast differentiation in mice, thus providing a new therapeutic approach for treating patients with osteoporosis.

Cbln1 and Cbln4 Are Structurally Similar but Differ in GluD2 Binding Interactions.

Unlike cerebellin 1 (Cbln1), which bridges neurexin (Nrxn) receptors and δ-type glutamate receptors in a trans-synaptic triad, Cbln4 was reported to have no or weak binding for the receptors despite sharing ∼70% sequence identity with Cbln1. Here, we report crystal structures of the homotrimers of the C1q domain of Cbln1 and Cbln4 at 2.2 and 2.3 Å resolution, respectively. Comparison of the structures suggests that the difference between Cbln1 and Cbln4 in GluD2 binding might be because of their sequence and structural divergence in loop CD. Surprisingly, we show that Cbln4 binds to Nrxn1ß and forms a stable complex with the laminin, nectin, sex-hormone binding globulin (LNS) domain of Nrxn1ß. Furthermore, the negative-stain electron microscopy reconstruction of hexameric full-length Cbln1 at 13 Å resolution and that of the Cbln4/Nrxn1ß complex at 19 Å resolution suggest that Nrxn1ß binds to the N-terminal region of Cbln4, probably through strand ß10 of the S4 insert.

Molecular basis for the recognition of CCN1 by monoclonal antibody 093G9.

CCN1, also named Cyr61 (cysteine-rich protein 61), is the first identified member of the CCN family that is composed of 6 secreted extracellular matrix-associated glycoproteins. CCN1 has been demonstrated to participate in pathogenesis of rheumatoid arthritis through various pathways. A monoclonal antibody, namely, 093G9, is effective to antagonize the effects of CCN1 and hence has potential therapeutic benefits against rheumatoid arthritis. Here, we show that the epitope recognized by 093G9 is mapped to residues 77 to 80 of CCN1, and a cyclic peptide encompassing residues 75 to 81 of CCN1 displays high binding affinity for 093G9. The crystal structure of the 093G9 Fab in complex with the cyclic peptide was determined at 2.7 Å resolution, which reveals the intensive interactions between CCN1 and 093G9. Particularly, residues Asn79 and Phe80 of CCN1 are inserted into cavities mainly formed by residues of complementarity-determining region loop L3 and framework region L2 and by residues of complementarity-determining region loops H2 and H3, respectively, which contribute most of the interactions and therefore are critical for the recognition by 093G9. Together, these findings not only identify the epitope of CCN1 for 093G9 but also reveal the molecular mechanism of recognition and binding of CCN1 by 093G9.

Regulation of Lrp6 phosphorylation.

The Wnt/beta-catenin signaling pathway plays important roles in embryonic development and tissue homeostasis, and is implicated in human disease. Wnts transduce signals via transmembrane receptors of the Frizzled (Fzd/Fz) family and the low density lipoprotein receptor-related protein 5/6 (Lrp5/6). A key mechanism in their signal transduction is that Wnts induce Lrp6 signalosomes, which become phosphorylated at multiple conserved sites, notably at PPSPXS motifs. Lrp6 phosphorylation is crucial to beta-catenin stabilization and pathway activation by promoting Axin and Gsk3 recruitment to phosphorylated sites. Here, we summarize how proline-directed kinases (Gsk3, PKA, Pftk1, Grk5/6) and non-proline-directed kinases (CK1 family) act upon Lrp6, how the phosphorylation is regulated by ligand binding and mitosis, and how Lrp6 phosphorylation leads to beta-catenin stabilization.

Cell cycle control of wnt receptor activation.

Low-density lipoprotein receptor related proteins 5 and 6 (LRP5/6) are transmembrane receptors that initiate Wnt/beta-catenin signaling. Phosphorylation of PPPSP motifs in the LRP6 cytoplasmic domain is crucial for signal transduction. Using a kinome-wide RNAi screen, we show that PPPSP phosphorylation requires the Drosophila Cyclin-dependent kinase (CDK) L63. L63 and its vertebrate homolog PFTK are regulated by the membrane tethered G2/M Cyclin, Cyclin Y, which mediates binding to and phosphorylation of LRP6. As a consequence, LRP6 phosphorylation and Wnt/beta-catenin signaling are under cell cycle control and peak at G2/M phase; knockdown of the mitotic regulator CDC25/string, which results in G2/M arrest, enhances Wnt signaling in a Cyclin Y-dependent manner. In Xenopus embryos, Cyclin Y is required in vivo for LRP6 phosphorylation, maternal Wnt signaling, and Wnt-dependent anteroposterior embryonic patterning. G2/M priming of LRP6 by a Cyclin/CDK complex introduces an unexpected new layer of regulation of Wnt signaling.

Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction.

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.